Dissection of the AIRE promoter region and its potential role in the pathogenesis of alopecia areata
Auteur : Dr. Rachid TAZI-AHNINI (Biomedical Genetics – Division of Genomic Medicine – University of Sheffield Medical School – The Royal Hallamshire Hospital – Beech Hill Road – Sheffield S10 2RX ENGLAND).
Travail subventionné par l’AAA (subvention 2005) à hauteur de 7 600 €.
Subvention débloquée le 28 janvier 2006.
Compte rendu fourni le 20 mars 2007.
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Summary of work – AIRE promoter 2005
Screening for single nucleotide polymorphisms (SNPs)
We used WAVE denaturing HPLC to screen this 600bp region in 64 individual DNA samples for the presence of any SNP’s within AIRE promoter region. We detected two SNPs with the AIRE promoter region at positions –103 and –528 respectively (see figure 1).
We have developed assays for each of the AIRE –528 and –103 polymorphisms. We did genotyped the AIRE –528 and –103 polymorphisms in 172 and 118 matched controls. Allelic discrimination analysis shows that neither the AIRE –103 nor the AIRE –528 polymorphism is significantly associated with alopecia areata.
Reporter gene assay
In preparation for reporter gene assays, different promoter haplotypes have been cloned and then sub-cloned into the pGL3-basic reporter vector. We are only considering SNP’s that have been validated for the different haplotypes, and at present have constructs with the following haplotypes; -528G/-103C, -528G/-103T and -528A/-103C. A negative control construct has also been produced. It contains the -528G allele, but will have the region -315 to -77 removed, as this is most of the minimal promoter region, but the construct stills contain the TATA box so that a basal level of transcription can still occur. These different promoter haplotypes have been sub-cloned with the firefly luciferase gene into a pcDNA5/FRT expression vector that had its Human Cytomegalovirus (CMV) immediate early promoter removed to generate pcDNA5/FRT-ApFL.
Transfection of TEC 1A3 cell line with promoter constructs
A human thymic epithelial cell line (TEC 1A3) has been transfected with pFRT/lacZeo and transfectants with pFRT/lacZeo successfully integrated into the cell lines genome were selected for using Zeocin. Seven to ten separate clones from successful transfectants have been isolated and maintained with the aim of extracting genomic DNA for use in Southern blot to screen cell lines that contain only a single copy of pFRT/lacZeo. Subsequently, single integrant cell lines will then be screened for transcriptional activity from the inserted plasmid vector using a b-galactosidase activity assay. The cell line with sufficiently high transcriptional activity will be used for transfections with pcDNA5/FRT-ApFL constructs of each different AIRE promoter haplotype. The resultant cell lines will all be identical, except for AIRE promoter identity, and will be assayed for firefly luciferase activity, with the intent that any differences between cell lines will be due to differences in the AIRE promoters.
We have identified two SNPs within the AIRE gene at positions -528 and -103 within the promoter region. We did no find any significant association between the AIRE –528 or –103 and alopecia areata in our cohort. In addition, three AIRE promoter haplotypes containing the AIRE –528 and –103 variants were cloned in pFRT/lacZeo and used to transfect TEC 1A3 cells. We are currently screening these clones for single integrant to be able to compare the transcription activity of these AIRE promoter haplotypes.
Figure 1. Wave dHPLC trace showing samples containing novel SNP, -528A, along with samples lacking this SNP.